Because cartilage tissue is avascular tissue and the ratio of cells in the tissue is very low, spontaneous regeneration is very limited. After Brittberg et al.'s report (1994) about autologous articular chondrocyte implantation (ACI) in which autologous articular chondrocytes of patients are isolated and proliferated, and then implanted into cartilage damaged area, ACI has been used for the treatment of articular cartilage damage and successful results have been reported in long-term observation. However, in cases of elderly patients and large-size damage, structural features or constitutions of normal articular cartilage cannot be reproduced. In the limits of conventional ACI (first-generation ACI), the following have been indicated as causes: considerably invasive implantation method in which cells in suspension are fixed at damage site with periosteum as a cover, the decreased viability of cells and non-maintenance of phenotype of chondrocytes, and weak physical strength. To overcome such limits, tissue-engineered cartilages—which are second-generation ACI technique using gel, membrane or three-dimensional scaffold as a cell carrier system—have been developed (Hutmacher et al., 2000; Adkisson et al., 2001; Ochi et al., 2001; and Cancedda et al., 2003).
In the preparation of tissue-engineered cartilages, a scaffold provides chondrocytes with three-dimensional system to maintain phenotype of chondrocytes and promote the production of hyaline cartilaginous extracellular matrix (ECM). In addition, the scaffold carries cells to cartilage-damage site and protects cells from loaded force by providing physical support at the implantation site. At present, scaffolds for tissue engineering have been developed by the use of many synthetic or natural materials. However, in view of clinical application, xenogeneic and allogeneic natural materials may cause immune reaction, and in the case of synthetic materials safety problems may be caused due to the harmful degradation product. Furthermore, when chondrocytes are inoculated to a scaffold, most of them distribute to the outer part of the scaffold, and extracellular matrixes which are synthesized and secreted by chondrocytes form shell at the outer part of the scaffold to hinder diffusion and exchange of nutrients, wastes and gases, resulting in death of interior cells. In some studies, successful tissue-engineered cartilages have been made by the use of scaffold. However, there are still many unresolved problems such as interaction between cells and biomaterials, irregular degradability of biomaterials, biocompatibility, uneven cellular distribution, lack of linkage (bonding) between tissue-engineered cartilage and peripheral cartilage and the like (Sittinger et al., 1996; Grande et al., 1997; Nehrer et al., 1998; Sims et al., 1998; Hutmacher et al., 2000; Ochi et al., 2001; Naumann et al., 2004; Park et al., 2006; and Wolf et al., 2008).
Studies about methods for the preparation of three-dimensional cartilage tissue without the use of scaffold have been continually carried out, but it has been reported that such methods are very limited in direct clinical applications since tissues are formed depending on only cells and ECM synthesis capacity of cells so that it is difficult to prepare tissues suitable for the size of damage where implantation is needed (Adkisson et al., 2001; Grogan et al., 2003; and Marlovits et al., 2003). Because cartilage is avascular tissue, it endures hypoxia and undernutrition well. However, Jain et al. (2005) and Rouwkema et al. (2008) state that because all cells in the body are not distant from blood vessels beyond 100 to 200 μm, when tissues for implantation are prepared at laboratories their size should be determined in view of limited nutrients, and diffusion of wastes and gases. In addition, the shape and depth of damaged cartilage are not uniform (FIG. 1A). Therefore, if three-dimensional cartilages prepared at laboratories are larger than the damaged area, implants should be trimmed in accordance with shapes of damages. On the contrary, if cartilage implants are smaller than the damaged area, implantation should be carried out in the manner of putting the pieces as a mosaic in accordance with shapes of damages. Tissue-engineered cartilages developed up to now are implanted in such a manner, but they cannot be adjusted to the thickness of damage. In such a case, at articular cartilages if implants highly protrude or are dented in comparison with adjacent cartilages, additional damages may be caused to implants or adjacent normal cartilage due to abnormal weight load (FIG. 1B).
Therefore, if small bead-type cartilage tissue is prepared, the death of interior cells—which is caused by the problem of perfusion in the course of culture—does not occur, and damaged areas can be restored regardless of the shape and thickness of cartilage-damaged area by inserting several small bead-type tissues into the damaged area. In addition, implantation can be performed by injecting into the damaged area via small incision or an arthroscope without large incision (FIG. 1C). However, for development as a therapeutic agent, a technique for preparing uniform cartilaginous tissues with repetitive reproducibility is necessary, and a large-scale culture system for preparing considerably large number of cartilaginous tissues is required for using in wide damaged area.
Methods for the preparation of three-dimensional hyaline cartilaginous tissue without the use of scaffold are based on the high-density three-dimensional culture of chondrocytes or cells having chondrogenic potential, and the maintenance of three-dimensional state in high density is the most important factor to express the phenotype of chondrocytes. At the development stage, after aggregation of chondroprogenitor cells chondrogenic differentiation is facilitated by the increase of initial cell-cell and cell-substrate adhesion molecules (Tavella et al., 1997; Stewart et al., 2000; Anderer et al., 2002; and Zhang et al., 2004).
Among methods in which small cartilaginous structures without a scaffold are prepared, first of all a pellet culture is a method in which from the initial step of three-dimensional culture an ultra-high-density culture system of cells is artificially made by the use of cell condensation which is prepared by centrifuging a considerably small number of cells. A pellet-formation procedure is simple and easily reproducible, and cells having chondrogenic potential make cartilaginous tissues by synthesizing and secreting cartilaginous matrix under this system (Zhang et al., 2004). A pellet culture method is the most frequently used method to evaluate chondrogenic potential of stem cells (Pittenger et al., 1999), and is also used for evaluating effects of external factors on chondrocytes (Croucher et al., 2000; Graff et al., 2000; Stewart et al., 2000; and Larson et al., 2002). However, evaluation of applicability of cartilaginous structure prepared by pellet culture as a cell-therapy product has not been carried out since a pellet system is a useful method for preparing high quality cartilaginous tissues but it has been regarded being difficult to apply to regeneration of damaged cartilage for the problem of difficulty in preparing sufficient pellet size. In addition, a general pellet culture uses a method in which cell suspension is added to a tube with a lid (a conical tube, a storage tube, a microcentrifuge tube and the like) and centrifuged, and three-dimensional culture is then carried out so that it can prepare only one pellet per one tube. As a result, it is difficult to apply this method to large-scale culture (FIG. 2A).
As methods for preparing small cartilage structure without a scaffold, there is a method to induce aggregation of cells spontaneously. Moscona et al. (1961) prepared a cell aggregate named as “aggregation pattern” by the use of rotation technique. They reported that when dynamic culture of cells suspended in a culture medium is carried out, a cell aggregate is spontaneously formed via interaction between cells (FIG. 2B). Landry et al. (1985; 1984) operated with cells on a non-adherent plastic substratum to prepare a three-dimensional cell aggregate and named it as “spheroid.” Reginato et al. (1994), Stewart et al. (2000), Anderer et al. (2000) and Wolf et al. (2008) induced the formation of spontaneous cell aggregate by culturing chondrocytes in a non-adherent culture dish coated with agarose or hydrogel (FIG. 2C). In such spontaneous spheroid system, cells form a three-dimensional cell aggregate and produce their own extracellular matrix (ECM) which is similar to natural matrix of hyaline cartilage. However, this culture method cannot adjust the number of cells which produce one cell aggregate, and there is a disadvantage of not being standardized as a tissue-engineered/cell therapy product since the size of each cartilaginous tissue and chondrification vary due to the possibility of fusion between formed cartilaginous tissues.
As another method to induce aggregation of cells spontaneously, there is a method uses an adherent culture dish. Imabayashi et al. (2003) placed drops of high-concentration suspension of cells having chondrogenic potential on an adherent culture dish and kept it in a 37° C. incubator. After several hours or days, cells were aggregated, and this aggregate was suspended in culture medium and three-dimensional culture was then carried out in a non-adherent culture dish or dynamic culture condition (FIG. 2D). This method, known as micromass/chondrosphere culture, has an advantage in that the number of cells forming cartilaginous tissue can be adjusted. However, it cannot be guaranteed to stably obtain uniform cartilaginous tissues since the capacity of forming cell aggregate spontaneously is different depending on cell condition. In addition, if cell aggregates are cultured all together before hardening of ECM, fusion between cartilaginous tissues may occur.
To equalize the number of cells forming cell aggregate spontaneously, studies using microwells have been conducted. In the three-dimensional culture of hepatocytes, if the hepatosphere is large, necrosis of internal core may occur. As a result, there is a need to develop three-dimensional culture system capable of preparing large amounts of uniform hepatospheres with a desired size. Fukuta et al. (2006) developed a method like to micromolding techniques as one of such methods. Wong et al. (2011) and Choi et al. (2010) prepared concave micromolds with 300-500 μm diameters based on thin poly-dimethylsiloxane (PDMS) membrane. They reported that when hepatocytes are cultured on plane PDMS surface, or in cylindrical or concave microwells to form spheroids, the size and shape of spheres formed in concave microwells were uniform; their size was perfectly regulated by the diameter of the concave microwells; cells cultured in concave microwells formed spheres more rapidly than those cultured in cylindrical microwells or on planar surfaces; and the spheres formed in concave microwells were easily harvested, which was a great advantage for generating stable spheres (FIG. 2E). After commercialization of molds, micro-tissue preparation methods using micromolds have been evaluated in various cells. However, because they are also methods to induce spontaneous cellular aggregation, it cannot be guaranteed that they stably obtain uniform cartilaginous tissues. In addition, because the size of prepared cell aggregates is too small, physical strength is weak and handling is difficult, so there is a limit in it being used as a therapeutic agent of three-dimensional chondrocyte.
As such, methods known up to now as those for preparing three-dimensional cartilage tissue without the use of scaffold have problems such that sufficient pellet size is not formed, it is not suitable for large-scale preparation, uniform cartilage tissue with repetitive reproducibility is not formed, the size of formed cell aggregate is too small, and strength is low. Therefore, such methods are inappropriate for the preparation of a therapeutic agent of bead-type chondrocyte without a scaffold